Practical context of enzymatic treatment for wound healing: A secreted protease approach (Review)
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BIOMEDICAL REPORTS 13: 3-14, 2020
Practical context of enzymatic treatment for wound
healing: A secreted protease approach (Review)
MARÍA ISABELA AVILA‑RODRÍGUEZ1, DAVID MELÉNDEZ‑MARTÍNEZ1, CUAUHTEMOC LICONA‑CASSANI1,
JOSÉ MANUEL AGUILAR‑YAÑEZ1,2, JORGE BENAVIDES1 and MIRNA LORENA SÁNCHEZ3
1
Tecnologico de Monterrey, Escuela de Ingeniería y Ciencias, Monterrey, Nuevo León 64849; 2Scicore Medical SAPI de CV,
Monterrey, Nuevo León 64920, Mexico; 3Laboratorio de Materiales Biotecnológicos, Departamento de Ciencia y Tecnología,
Universidad Nacional de Quilmes-Imbice-Conicet-Cicpba, Bernal, Buenos Aires B1876BXD, Argentina
Received October 1, 2019; Accepted February 14, 2020
DOI: 10.3892/br.2020.1300
Abstract. Skin wounds have been extensively studied as their for use, sources or types of proteases used for wound healing
healing represents a critical step towards achieving homeo- have been found in the literature to date. Through the present
stasis following a traumatic event. Dependent on the severity of review, the context of enzymatic wound care alternatives will
the damage, wounds are categorized as either acute or chronic. be discussed. In addition, substrate homology of SPs and
To date, chronic wounds have the highest economic impact human MMPs will be compared and contrasted. The purpose
as long term increases wound care costs. Chronic wounds of these discussions is to identify and propose the stages of
affect 6.5 million patients in the United States with an annual wound healing in which SPs may be used as therapeutic agents
estimated expense of $25 billion for the health care system. to improve the wound healing process.
Among wound treatment categories, active wound care repre-
sents the fastest‑growing category due to its specific actions
and lower costs. Within this category, proteases from various Contents
sources have been used as successful agents in debridement
wound care. The wound healing process is predominantly 1. Introduction
mediated by matrix metalloproteinases (MMPs) that, when 2. Chronic wound healing management: Practical context of
dysregulated, result in defective wound healing. Therapeutic traditional and enzymatically based debridement approaches
activity has been described for animal secretions including fish 3. MMPs in skin wound healing: Comparison and substrate
epithelial mucus, maggot secretory products and snake venom, homology with proteases secreted from other animals
which contain secreted proteases (SPs). No further alternatives 4. Potential of SPs as novel alternatives for wound healing care
5. Future perspectives
Correspondence to: Dr Mirna Lorena Sánchez, Laboratorio de 1. Introduction
Materiales Biotecnológicos, Departamento de Ciencia y Tecnología,
Universidad Nacional de Quilmes-Imbice-Conicet-Cicpba, Roque A wound of the skin is generally described as the interrup-
Sáenz Peña 352, Bernal, Buenos Aires B1876BXD, Argentina tion of the epithelial surface caused by a physical or thermal
E‑mail: mirna.sanchez@unq.edu.ar challenge (1). Skin wounds have been extensively studied as
Dr Jorge Benavides, Tecnologico de Monterrey, Escuela de Ingeniería their healing represents a critical step in achieving homeostasis
y Ciencias, Av. Eugenio Garza Sada 2501 sur, Monterrey, Nuevo following a traumatic event. Dependent on the severity of the
León 64849, México damage, wounds are categorized into either acute or chronic (2).
E-mail: jorben@tec.mx To date, chronic wounds have the highest economic impact as
long term treatment increases wound care costs (3). It is esti-
Abbreviations: ECM, extracellular matrix; FMC, fish mucus mated that 1‑2% of the population of the developing world will
cathepsin; FMM, fish mucus meprins; FMMPs, fish matrix
experience a chronic wound in their lifetime (4). According to
metalloproteinases; FMSP, fish mucus serine proteases; MaPs,
maggot proteases; MMPs, matrix metalloproteases; SPs, secreted
Brem et al (5), in 2007 chronic wounds had affected 6.5 million
proteases; SVMPs, snake venom metalloproteases; SVSPs, snake patients in the United States, with an annual estimated health
venom serine proteinases; TGF‑β1, transforming growth factor‑β1; care expense of $25 billion (6). However, to date, the actual
VEGF, vascular endothelial growth factor cost of chronic wound care in the United States is unknown (7).
There has been a relatively high increase in the incidence of
Key words: enzymatic wound treatment, fish epithelial mucus, chronic wounds, and this may be closely associated with
maggot secretory products, matrix metalloproteases, snake venom the increase in factors which impair wound healing, such as
proteases diabetes, obesity, or therapeutics such as chemotherapy, steroids
and non‑steroidal anti‑inflammatory drugs (6).4 AVILA‑RODRÍGUEZ et al: ENZYMATICALLY TREATED WOUND HEALING, A SECRETED PROTEASE APPROACH
The cost of chronic wound care represents a complicated This failure represents an important baseline to treat
scenario for patients and health care systems, leading to a chronic wounds, as devitalized epithelium builds up a physical
necessity for the development of healing solutions which are barrier that precludes the healing process by interfering
both quicker and more cost‑effective. To date, the available with the repair machinery, mimicking signs of infection,
wound treatment therapeutics are: dressings, such as antimi- providing nutrients to anaerobic pathogenic agents, such as
crobial, films and alginate; hydrocolloids, collagen products, Clostridium perfringens or Bacteroides sp., and promoting
gauze composites and hydrogels; and active wound care (8). cytokine production that in severe cases generates a septic
Active wound care represents the fastest growth category response (28).
(20.6% compound annual growth rate between 2016‑2022) Debridement can be performed through autolytic, surgical,
as it is an alternative that has a more specific action and is biological or enzymatic means (28). Of these, autolytic debride-
more cost‑effective (9). Within the active wound care category, ment is the most conservative treatment strategy. It enhances
proteases from a range of sources have been employed as the action of endogenous phagocytic cells and proteases such
successful agents in debridement (10), enhancing wound as MMPs through dressings that provide the ideal catalytic
healing (11), coagulation (12) and keloid scar treatments (13). conditions for removal of necrotic tissue (29). Among the
Of these, debridement comprises the principal dermato- dressings available for autolysis, films (polydimethylsiloxane),
logical application in enzymatic wound care, a proven and gauzes, hydrocolloids, hydrogels, alginates, hydrofibers and
well‑established principle (14). foams have been proposed (25,30). This strategy is selec-
The wound healing process is predominantly mediated by tive, painless, inexpensive and suitable for most types of
matrix metalloproteinases (MMPs) (15‑17). Dysregulation of wounds (31). However, this process is slow, dependent on
MMPs results in defective wound healing, which has made suitable reaction conditions and on the physiological response
them targets of study in cases of chronic wounds, diabetic foot of the patient, and carries the risk of skin degradation due to
injury, keloid healing and burned skin (10). The topical appli- prolonged exposure to moisture (maceration) (32) within the
cation of non‑human proteases has demonstrated beneficial surrounding skin (28).
therapeutic effects in events where MMPs fail due to dysregu- Surgical debridement strategies are performed by excising
lation, for example in hemostasis (18), wound closure (19) and necrotic tissue until only healthy skin regions are exposed (33).
debridement (20). Available variants of surgical debridement include ultrasound
Debridement is the most widely explored enzymatic wound debridement, plasma‑mediated bipolar radio‑frequency abla-
care application, in which the most frequently used proteases tion, versa‑jet (fluid jet technology) and hydrosurgery (34,35).
are collagenases, serine proteases and cysteine proteases. The Surgical debridement is the fastest and most effective route of
therapeutic activity of animal secretions from fish epithelial treatment, but is an expensive method that requires a sterile
mucus (21), maggot (Lucilia sericata) secretory products (22) surgical environment, trained practitioners, and specific
and snake venom (23) have also been demonstrated. These instruments, and is contraindicated for patients with clotting
secretions contain different types of proteases capable of disorders (28,36).
degrading the same substrates as MMPs. Besides these, no By contrast, biological debridement promotes the removal
further use cases, sources or types of proteases for wound of devitalized epithelium through the digestive action of
healing were found based on the currently available literature. Lucilia sericata sterile maggots (31). Maggots are caged
Through the present review, the context of enzymatic in wound‑sized hydrocolloid dressings that are placed in
wound care alternatives will be discussed, along with a the affected area (37). The secretion of several components
comparison of substrate homology of secreted proteases (SPs) including proteolytic enzymes, such as trypsin and chymo-
and human MMPs. This review will aid in the identification of trypsin serine proteases, then catalyze non‑viable skin into a
which stages of the wound healing process SPs may be used as liquid feedstock that facilitates maggot feed (38). This alter-
therapeutic agents. native has proved to be efficient in several types of chronic
wounds (39) and ulcers (40,41) by providing quick wound
2. Chronic wound healing management: Practical context debridement, reduction in the use of biofilms, disinfection from
of traditional and enzymatically based debridement bacteria (40,42‑45) and improved pain control (46). However,
approaches due to the negative image several societies impose on maggots,
this alternative has not been well accepted by patients and
Debridement is the first step to enhance repair of chronic practitioners (47). Furthermore, it is contraindicated for the
wounds. According to the European Wound Management treatment of fistulae, exposed vessels and wounds in proximity
Association, this procedure is considered a basic necessity to to vital organs (42).
induce the physiological process of tissue repair (24). Through A potential compromise is enzymatic debridement, in
debridement, necrotic tissue is removed by external means which proteases from different sources (bacterial, vegetal or
to create a stable and healthy scaffold for re‑epithelializa- animal) is applied to the wounded area to remove necrotic
tion (25). In healthy individuals under normal circumstances, tissue (48,49). Enzymatic debridement is selective and suit-
debridement is performed naturally following clot formation able for infected wounds (36), without the need for complex
by neutrophil‑derived MMPs and other components (26). equipment or application procedures. This alternative also
However, when the MMP machinery fails, there is an accumu- takes less time and requires fewer applications to accomplish
lation of devitalized tissue. As a consequence, the steadiness debridement compared with dressings used for autolytic
of prolonged catabolism diminishes re‑epithelialization and treatments (50). Other reported enzymatic wound healing
results in chronic wounding (27). approaches are anti‑ or pro‑coagulation through venom toxinsBIOMEDICAL REPORTS 13: 3-14, 2020 5
from Bothrops sp. (51,52). These enzymes may frequently The presence of SPs has been reported in the secretions of
be inhibited by salts, temperature and hydrogen peroxide, fish (70), maggots (71,72) and snake venom (73). As MMPs are
which are common elements of aseptic solutions. A stinging one of the primary participants of the wound healing process,
sensation and exudate may also be observed as an after‑effect a similarity may exist in the catalytic mechanisms of SPs and
of enzymatic treatment (36). MMPs. This similarity may explain the therapeutic effect
From these four mentioned alternatives, three are directly provided by these secretions.
dependent on proteases to perform the debriding activity. Maggot therapy efficiency in the treatment of necrotic,
The direct or indirect use of proteases is therefore the second infected chronic wounds is due to the activity of several SPs.
most commonly used tool after surgical debridement. In This secretion consists of serine proteases (trypsin‑like and
the current literature, the most commonly used proteases chymotrypsin‑like) and metalloproteases (71,72). As a secre-
in direct enzymatic debridement are bromelain, papain tion, maggot proteases (MaPs) contribute to the wound healing
and bacterial collagenases (53). Other enzymes have been process, primarily in fibroblast stimulation and bacterial
demonstrated to intervene as anti‑ or pro‑coagulation agents disinfection. MaPs degrade fibrin clots and fibronectin (74),
and in non‑specific wound healing from animal secretions. enhancing fibroblast metabolism and migration (22,75). In
The most common commercially and non‑commercially addition, MaPs increase TGF‑β (transforming growth factor‑β)
available proteases associated with wound healing are listed signaling in wounds treated with maggots (76), which enhances
in Table I. endothelial cell and keratinocyte migration, thus promoting
Animal secretions with high quantities of protease content, wound closure. Furthermore, MaPs inhibit neutrophil
including fish epithelial mucus and snake venom, have been migration and decrease the production of pro‑inflammatory
reported to enhance wound healing. Wound healing proper- mediators in neutrophils and monocytes (44,77), leading to
ties were reported for the secreted mucus of the fish species recruitment of pro‑angiogenic growth factors (78) and healthy
Netuma barba (54), Channa striatus (55) and Clarias gari- granulation tissue (79). MaPs are also considered antimicrobial
epinus (56). A reduction in healing time of almost 60% was enzymes (80), capable of eliminating Staphylococcus aureus
achieved following the topical application of mucus prepara- and Pseudomonas aeruginosa (44,81) as well as degradation
tions in the wounds of mice, rats, guinea pigs and humans (57). of biofilms produced by S. epidermidis and S. aureus (41).
For snake venom, anti‑ or pro‑coagulation and epithelial cell From MaPs, only a chymotrypsin‑like protease has been
migration properties were observed with the toxins from the isolated from maggot secretions, which exhibited clotting
venom of Bothrops moojeni, B. atrox (51), B. alternatus (18) and proteolytic activity in fibronectin, suggesting its use in
and B. jararaca (58). hemostasis and for temporary collagen‑rich replacement of
Thus far, the primary application of proteases in wound ECM (74,82). These proteases also reduce biofilms in patients
treatment has been debridement. Information regarding the use with leg ulcers (40,41).
of proteases being used for other wound healing treatments is Similar to maggot secretions, fish mucus and snake venom
scarce, suggesting that relatively little attempt has been made have been hypothesized as wound healing treatment agents.
to propose the use of proteases in different stages of the wound In traditional medicine, they have been used as a therapy for
healing process (57,59). Several therapeutic benefits have been skin burns and hemostasis (51,55,56,83). Fish epithelial mucus
described from animal secretions, but studies on their possible consists primarily of glycoproteins and immune biomol-
use in wound healing stages are limited. It may be beneficial to ecules (84). Immune components, metalloproteases, serine
determine whether the existing types of SPs present in animal proteases, and cathepsins B, D and L, have been identified in
secretions with reported therapeutic effects (maggots, fish and fish epithelial mucus (85,86). Enzymatic components from
snakes), can mimic human MMPs. crude secretions contribute to accelerated clot formation and
agglutination of red cells (87).
3. MMPs in skin wound healing: Comparison and substrate In the case of metalloproteases, fish matrix metallopro-
homology with proteases secreted from other animals teinases (FMMPs) 9 and 13 and fish mucus meprins (FMM)
have been described as components of fish mucosal secre-
Wound healing is the process by which an epithelial tions (88,89). FMMPs 9 and 13 have analogous variants in
discontinuity is closed and is divided into four major steps: human tissue, which participate in wound contraction and
Hemostasis, inflammation, cell migration‑proliferation and re‑epithelialization (66,90). FMMs can degrade collagen IV,
skin remodeling (60,61). The interaction and co‑ordination of fibrillar procollagen and fibronectin (91‑93), which are also
several elements such as cytokines, growth factors, coagulation degraded by MMPs 3, 10, 11 and 12 (Table II). These proteases
elements, extracellular matrix (ECM) components, paren- are involved in wound contraction, monocyte/macrophage
chymal cells and MMPs (62,63) enable the correct progression metabolism and re‑epithelialization (66).
of these major steps (Fig. 1). Cathepsins are a family of proteases that have been
It has been reported that MMPs predominantly mediate identified in fish epithelial mucus, and these cathepsins in
the wound healing process and are involved in several events fish mucus have not been characterized. It is hypothesized
in each stage, including ECM degradation (64), cell prolifera- that the cathepsins in fish mucus may exhibit a therapeutic
tion/migration, mesenchymal cell differentiation (65), wound effect on wound healing based on the available data regarding
contraction, angiogenesis and re‑epithelialization (66‑68). At their properties on human skin. These proteases are normally
present, 25 different MMP variants have been identified in the present in lysosomal vesicles, but their presence has also been
human genome (64,69). Of these, 11 are responsible for skin demonstrated extracellularly (94). In human physiology, they
remodeling and wound healing (Table II). participate in wound healing during hemostasis (95), ECM6 AVILA‑RODRÍGUEZ et al: ENZYMATICALLY TREATED WOUND HEALING, A SECRETED PROTEASE APPROACH
Table I. Applications of proteases in wound healing treatments classified by their reported therapeutic effect.
A, Debridement and skin burns
Author, year Enzyme Source (Refs.)
Ford et al, 2006 Papain + urea (Accuzyme SE) Carica papaya (152)
Ford et al, 2006 Papain, Urea, Chlorophyllin Copper (152)
Complex Sodium (Panafil SE)
Muhammad et al, 2014; Papain/Chymopapain (20,153)
Yaakobi et al, 2007
Klasen, 2000 Collagenase Clostridium sp. (14)
Smith & Nephew, Inc., 2014 Collagenase (Santyl )
®
C. histolyticum (154)
Giudice et al, 2017 Bromelain (NexoBrid) Ananas comosus (155)
Gorecki and Toren, 2005 Bromelain cysteine protease (156)
Klein and Houck, 1980 Bromelain cysteine protease (157)
Niehaus et al, 2012 Debrilase Lucilia sericata (158)
Niehaus et al, 2012 Serine protease (159)
Rosenberg, 2012 Bromelain, trypsin enzyme H‑4, collagenase, Several (160)
papain/papain‑urea
Freeman et al, 2012 Collagenase, elastase, papain, bromelain, (161)
hydrolase, streptokinase
B, Anticoagulation and procoagulation
Author, year Enzyme Source (Refs.)
Waheed et al, 2017 Moojenin (Defibrase®) Bothrops moojeni (51)
Waheed et al, 2017 Batroxobin (Reptilase) B. atrox (51)
Chan et al, 2016 Thromboplastin‑like and thrombin‑like (52)
(Hemocoagulase)
De Marco Almeida et al, 2015 Venom B. alternatus (18)
Yaakobi et al, 2004 Collagenase Non specified (162)
Rodeheaver et al, 1974 Trypsin/ADAMS SVMP Bovine (163)
Glyantsev et al, 1996 Collagenase Crab (specie non specified) (27)
Ferreira et al, 2017 Buffalo cryoprecipitate and Serine protease Crotalus durissus terrificus (59)
C, Enhancing wound healing
Author, year Enzyme Source (Refs.)
Fierro‑Arias et al, 2017 Collagenase C. histolyticum (13)
Gao et al, 2015 rMMP8 and MMP9 inhibitor Non specified (164)
Pasha et al, 2015 Cream/composite Channa striatus (143)
Rilley and Herman, 2005 Collagenase Clostridium sp. (19)
Ferreira et al, 2018 Jararhagin B. jararaca (58)
Mukherjee et al, 2017 Mucus Echinoida sp. (83)
Costa‑Neto, 2004 Globe eye Netuma barba (54)
Manan Mat Jais, 2007 Mucus C. striatus (55)
MMP, matrix metalloproteinase; SVMP snake venom metalloprotease; rMMP, recombinant MMP.
remodeling (96) and keratinocyte migration (97). Cathepsin‑L fibronectin, proteoglycans, and collagens I and II (100), while
substrate affinity has been described for laminins, fibronectin, substrate affinity of Cathepsin‑B has been described primarily
elastin and collagen (98,99). Cathepsin‑D has affinity for for collagen II, IX and XI (101). These substrates are alsoBIOMEDICAL REPORTS 13: 3-14, 2020 7
Figure 1. Simplified diagram of the interactions between different cell types during wound healing, the contribution of MMPs and proposed wound healing
mechanisms of SPs. Skin injury repair begins with hemostasis, a process which stops blood loss and provides a temporary matrix facilitating further steps in
wound healing. Fibrin‑rich ECM formation stimulates neutrophil‑activated monocyte recruitment through TNF‑α and PDGF. Both neutrophils and monocytes
produce several growth factors, such as TNF‑α, TGF‑α, TGF‑β, EGF and FGF, to enhance migration and proliferation of fibroblasts, endothelial cells, and
keratinocytes to the site of injury. Fibroblasts stimulate other cells to produce collagen deposits in the ECM, wound contraction, angiogenesis and re‑epitheli-
zation. Studies suggest that SPs, such as FMC, FMMP, FMM, FMSP, MaP, SVMP and SVSP, may behave similarly to endogenous MMPs during these stages.
Ang, angiopoietin; CTGF, connective tissue growth factor; Col, collagen; ECM, extracellular matrix; EGF, epidermal growth factor; FGF, fibroblast growth
factor; FMC, fish mucus cathepsin; FMMP, fish mucus matrix metalloprotease; FMM, fish mucus meprin; FMSP, fish mucus serine protease; FN, fibronectin;
Hy, hyaluronan; IL‑1, interleukin‑1; MaP, maggot protease; MMP, matrix metalloproteinase; PDGF, platelet‑derived growth factor; PG, proteoglycan; SVMP,
snake venom metalloprotease; SVSP, snake proteinase; TGF, transforming growth factor; TNF‑α, tumor necrosis factor‑α; VEGF, vascular endothelial growth
factor.
target proteins for MMPs 1, 8, 13 and 14 (66,102), which proteases: Snake venom metalloproteases (SVMPs) and snake
supports the reported role of cathepsins in wound contraction venom serine proteinases (SVSPs) (73). These enzymes cata-
and hemostasis. lyze a broad range of ECM components, coagulation factors
Additionally, fish mucus serine proteases (FMSPs) are and proteins involved in platelet aggregation (109,110).
present in mucosal secretions (103), albeit with only poor SVMPs can intervene in hemostasis, as these hydro-
substrate characterization thus far. Nevertheless, this family lyze glycoprotein Ib and factor X, which promote
of proteases has reported activity on collagen, elastin, fibrin coagulation (110‑112) and platelet aggregation (113,114),
and fibrinogen (104,105). Thus, this protease may be useful respectively. During inflammation, SVMPs enhance the infil-
during hemostasis, generating platelet aggregation and fibrin tration of inflammatory cells (115,116) as well as increasing
clot formation (106). Additionally, FMSPs degrade fibrin, neutrophil and macrophage numbers (117‑119), which
which may assist in the change of ECM from temporary increases soluble collagen levels and enhances angiogenesis
to collagen‑rich, resulting in cellular proliferation and through increasing vascular endothelial growth factor (VEGF)
migration (107). This family of enzymes also interferes and TGF‑β1 release (58). During cell migration and prolifera-
with the maturation of MMPs (66) and the desquamation tion, it has been demonstrated that SVMPs degrade fibrin and
processes (108). fibronectin (112,120), resulting in the change from temporary
Snake venoms, particularly from the Viperidae family, are to collagen‑rich ECM. SVMPs also activate migration of
rich in proteases. There secretion is comprised of two types of skin fibroblasts (121) and endothelial cells (111,122‑124). In8
Table II. Classification and function of human MMPs involved in skin remodeling and wound healing.
Family Type Function Source Substrates Dysregulation effects (Refs.)
Collagenases 1 Promotes re‑epithelialization Interstitial Collagen I, II and III In high levels generates (66,165‑171)
when cleaving native col 1 fibroblasts chronic wounds
8 Regulation of neutrophil Neutrophils Increased levels fibroblast
chemotaxis and effectors of lack apoptosis
inflammatory process
13 Maturation of granular tissue Stromal fibroblasts Collagen I, II, III, V Leads to arthritis, fibrosis,
and wound closure Human and XI atherosclerosis and cancer
chondrocytes
Gelatinases 2 Cleaves γ2 of laminin 332 Fibroblasts, Collagen (IV, I), Chronic wounds when (66‑68,102,
promoting keratinocyte endo thelial cells γ2 laminin 332, MMP‑2 is in high levels 170,172‑175)
migration, angiogenesis regulation alveolar epithelial gelatin
by p10 angiogenic cytokines cells
9 Keratinocytes Elastin, aggrecan, Wound closure impaired
fibronectin in MMP9 ‑/‑
and vitronectin
Stromelysins 3 Regulates wound healing (wound Dermal fibroblasts Collagen (II, III, IV, Increased expression has (66‑68,102,
contraction), activate pro‑MMPs and basal IX, X) proteoglycans, been reported in dystrophic 170,172‑175)
and releases bioactive cytokines keratinocytes laminin and fibronectin epidermolysis bullosa
(HB‑EGF, FGF)
10 Enhance migrating cell front Colocalized with Collagen III, IV and V Disorganized cell
in keratinocytes MMP1 in leading migration, degradation of
edge of the wound new matrix, aberrant cell to
keratinocytes cell contact and increase in
cell death of wound edge
11 Activation of pro‑MMPs Peritumoral α‑I‑antiprotease In increase expression
antiapoptotic fibroblasts collagen VI promotes tumor
development
Matrylisins 7 Wound re‑epithelialization and Stromal fibroblasts Pro‑MMP‑1, gelatin, Innate immunity (19,176‑178)
neutrophil migration enhancing in mucosal epithelia collagens defects decreased
through chemokine processing. re‑epithelialization
in lung injury
AVILA‑RODRÍGUEZ et al: ENZYMATICALLY TREATED WOUND HEALING, A SECRETED PROTEASE APPROACH
Membrane 14 Regulates epithelial cell prolif Cell membrane of Collagen (I, II, III), Defective collagen I (66,102)
bound eration by altering KFG receptor keratinocytes of the gelatin, fibronectin, production, loss of MMP2
and activates pro MMP2 migrating front laminin and impaired wound
healingBIOMEDICAL REPORTS 13: 3-14, 2020 9
addition, SVSPs exhibit proteolytic activity on Factor V and
fibrinogen, promoting fibrin clot formation (125‑127). SVSPs
(173,177,
179‑182)
(Refs.)
also promote aggregation of platelets (128).
Following analysis of reported interventions of SPs in
wound healing, it could be presumed that they can intervene
as helpers in several intermediate steps of the wound healing
processes including coagulation, ECM degradation for
re‑epithelialization, or wound contraction, among other steps.
The hypothesized mechanisms of SPs during the process of
Dysregulation effects
Increased angiogenesis
because of decreased wound healing are presented in Fig. 1. Study of these variants
may assist in the development of novel specific alternatives for
active chronic wound healing care.
angiotensin
4. Potential of SPs as novel alternatives for wound healing
proteoglycan
care
Substrate homology analysis among MMPs and SPs suggest
MMP, metalloproteinase; HB‑EGF, Heparin‑binding EGF‑like growth factor; FGF, fibroblast growth factor; KFG, KGF, Keratinocyte growth factor.
that animal enzymes may act similarly to the ones physiologi-
cally present in human skin. As presented in Fig. 1, previously
compared SPs may be used to facilitate several steps involved
laminin, vitronectin,
in the process of wound healing, or to compensate for the
elastin, fibronectin,
physiological variants when they do not function properly. To
Collagen (1, IV),
Substrates
understand this from a clearer perspective, it is important to
comprehend in which of the most common chronic wounds
types SPs may serve as suitable co‑adjuvants.
In the current literature, chronic wounds have been classified
into pressure ulcers, venous ulcers or diabetic ulcers (129,130).
Pressure ulcers are caused by pressure, shear force, friction or
a combination of these (131). The prevention and cure of pres-
sure ulcers is associated with daily movement of extremities
and frequent body positioning during hospitalization (132). In
this case, the use of proteases may serve as palliative care in
Source
Macrophages
bed preparation for wounded patients as opposed to assisting
the metabolic processes of wound healing.
Chronic venous ulcers are associated with inflamma-
tion, mechanical damage and erratic structural remodeling
microphage migration
of the vein. Pathological hemodynamics results in changes
to microcirculation; this produces thrombosis, proinflam-
matory activity and impaired MMP‑3 activity (133), leading
to cell dysfunction and finally to ulceration (134). For
ulceration and potential necrosis, maggot therapy has shown
efficacy (40,41) by decreasing inflammation and neutrophil
migration (77,135). It also degrades eschar, debrides the wound
Elastin degradation and
Function
and serves as a bacterial disinfectant (40,42‑45). Furthermore,
fish mucus proteases have been shown to exhibit antibacterial
activity (55,136), which may be useful for bacterial disinfec-
tion of ulcers.
Diabetic foot ulcers are wounds that manifest after a
cascade of metabolic dysregulations initiated by long‑term
hyperglycemia (137). As a result of prolonged exposure to high
blood sugar levels, there is a decrease in fibrinolytic activity,
thus increasing blood viscosity and coagulation in this type of
wound (138). In addition, hyperglycemia results in a reduction
Type
12
of growth factors and receptor levels (such as TGF‑β1), accom-
panied by a prolonged inflammatory phase due to upregulation
Table II. Continued.
of MMP‑9 (139,140), which interrupts the inflammatory and
proliferative phases of wound healing (141).
Other MMPs
As an alternative therapy for diabetic foot ulcers, maggot
treatment has demonstrated improved efficacy and efficiency
Family
compared with conventional methods (142). Furthermore,10 AVILA‑RODRÍGUEZ et al: ENZYMATICALLY TREATED WOUND HEALING, A SECRETED PROTEASE APPROACH
MaPs (74), FMMPs (91) and a certain type of SVMP (112,120) and instead use only the SPs. This may also allow heterologous
have been reported to exhibit fibrinolytic activity which may production, immobilization or improvement of the therapeutic
ameliorate the characteristic viscosity of diabetic ulcers. properties of the characterized SPs through mutagenesis. In
Additionally, it has been reported that TGF‑β signaling is addition, time‑efficient diagnostic tests on for detection of
increased in the presence of MaPs (76) and SVMPs (58), and this molecular targets in skin wound healing may be developed
may also assist wound healing in this type of ulcer. However, to guide practitioners on which tool to use for chronic wound
certain SVMPs can promote coagulation (110‑112,120); thus, care, resulting in improved wound healing and thus restoration
meticulous care must be taken to separate and study each of homeostasis.
component embedded within the secretion instead of applying
it as a whole. Acknowledgements
In another report, fish mucus application enhanced the
healing of laparotomy wounds (143). Therefore, SPs may be Not applicable.
used to reduce the time taken for wound healing or for the
removal of necrotic tissue, depending on the wound patho- Funding
physiology.
Despite the positive effects of SPs in wound healing, further The present study was funded by CONACYT (grant nos. 886264
research must be performed to determine the specific mecha- and 548216).
nisms of action, regulation, site delivery and bioavailability
of proposed proteases before they may be recommended as Availability of data and materials
feasible pharmacological candidates for treatment of chronic
wounds. The application of SPs may be limited however, as Not applicable.
its use for treatment of burn wounds exhibits highly variable
results in patients (14). Authors' contributions
It is also important to determine how SPs may affect
other wound healing mechanisms when used as an adjuvant All authors (MIAR, DMM, CLC, JMAY, JB and MLS)
with other healing methods such as skin transplants. In this contributed to writing, editing and revising the manuscript. All
procedure, lost skin is covered with healthy tissue or artificial authors approved the final version of the manuscript.
composites (144,145) that provide the necessary elements
(cells, growth factors, MMPs and scaffolds) for the healing Ethics approval and consent to participate
process (146). The success of a skin transplant is primarily
dependent on angiogenesis between the skin graft and the Not applicable.
injury, which is predominantly mediated by MMP‑2, 9 and
14 (147). Thus, SPs have been proposed as potential adjuvants Patient consent for publication
to increase tissue compatibility during skin transplants.
Nevertheless, studies on SP‑aided transplants is still Not applicable.
ambiguous. For example, the use of botulinum toxin A during
skin transplantation in murine models enhances the expression Competing interests
of VEGF and prolonged the survival of skin grafts (148). By
contrast, Kucukkaya et al (149) demonstrated that the same The authors declare that they have no competing interests.
toxin reduces wound‑graft contraction. Thus, the effects of SPs
on skin transplants requires additional studies to determine its References
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